US5635347A - Rapid assays for amplification products - Google Patents
Rapid assays for amplification products Download PDFInfo
- Publication number
- US5635347A US5635347A US08/188,943 US18894394A US5635347A US 5635347 A US5635347 A US 5635347A US 18894394 A US18894394 A US 18894394A US 5635347 A US5635347 A US 5635347A
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- US
- United States
- Prior art keywords
- nucleic acid
- acid sequence
- amplification product
- labeled
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
- G01N2458/30—Electrochemically active labels
Definitions
- This invention relates to the detection or quantitation of amplified nucleic acid sequences. More specifically, this invention relates to the detection or quantitation of nucleic acid analytes of interest in the amplification product of a polymerase chain reaction in rapid, one step, homogeneous assays.
- nucleic acid hybridization methods to detect disease states and infectious agents is a rapidly emerging technology (1). These methods have largely involved simple nonradioactive formats, aimed at achieving acceptance in the clinical laboratory (2,3). These assays use chemiluminescent or enzyme labels, or combinations of these to obtain the desired sensitivity (4).
- the rapid sample preparation offered by PCR offers the possibility of developing rapid and simple assays.
- a nucleic acid such as deoxyribonucleic acid (DNA) is able to serve as its own template during self-replication. It is also well known that a double stranded or duplex nucleic acid can be separated into its component single strands. These properties have been exploited to permit the in vitro amplification and modification of nucleic acid sequences by the polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- PCR is an in vitro, enzyme-based replication of nucleic acid sequences, using two oligonucleotide primers designed to hybridize to opposite strands and flank the region of interest on the target polynucleotide sequence.
- the nucleic acid is subjected to strand separation, typically by thermal denaturation, the primers are hybridized (by annealing if thermal cycling is used) to the single strand templates, and an enzyme such as DNA polymerase (DNA template to DNA primer extension) or reverse transcriptase (ribonucleic acid or "RNA" template to DNA primer extension or DNA template to DNA primer extension) extends the primers on the templates.
- DNA polymerase DNA template to DNA primer extension
- reverse transcriptase ribonucleic acid or "RNA" template to DNA primer extension or DNA template to DNA primer extension
- Both of the strands (plus and minus), including newly synthesized strands are made available as templates for the extension of both primers respectively by the strand separation step.
- the nucleic acid duplex which results will have termini corresponding to the ends of the specific primers used. It is possible, by means of PCR, to amplify, detect, or otherwise modify a nucleic acid sequence in vitro.
- primers for PCR require that the terminal sequences of the nucleic acid strands (both the plus and minus templates) to be amplified or detected, be known (5).
- the sequence information may be derived by direct sequencing of the terminals of the nucleic acid of interest, or by sequencing the terminal of a polypeptide and producing a corresponding copy oligonucleotide primer.
- the optimal primer size is typically about 20-30 bases in length, but workable primers may be smaller or larger in particular circumstances. As is well known, as primer size decreases, the likelihood that the primer will hybridize to an unplanned site on the sequence of interest increases. Unplanned hybridizations can lead to an interruption of amplification of the desired product and production of products having either a smaller size or an undesired primer insert. Thus, the selection of two optimal primers for PCR requires the avoidance of unplanned hybridization with the sequence of interest whenever practical.
- primer sequence to avoid unplanned hybridizations is well known. Algorithms are known by which the artisan may compare proposed primer sequences to the entire template sequence (where known) and to any other sequences which are known to be present in an assay mixture.
- PCR amplification has been used to isolate new gene sequences from a polynucleotide sequence library. While new genes may also be isolated by means of a sufficiently complementary probe incorporating a portion of the sequence of the new gene, such probe isolation methods lack the sensitivity provided by PCR.
- nucleic acid probes labeled at their 5' end have been used to hybridize to the nucleic acid analytes of interest. These probes occasionally enter into the polymerase chain reaction at their unlabeled 3' end resulting in spurious results.
- the prior art assays are also heterogeneous, i.e. separation assays. As such they are time consuming and the multiple wash steps involved introduce the possibility of contamination of the assay sample.
- nucleic acid analytes of interest Numerous methods and systems have been developed for the detection and quantitation of nucleic acid analytes of interest in biochemical and biological substances. Typically, the existence of a nucleic acid analyte of interest is indicated by the presence or absence of an observable "label" attached to a probe which binds to the analyte of interest. Of particular interest are labels which can be made to luminesce through photochemical, chemical, and electrochemical means.
- Photoluminescence is the process whereby a material is induced to luminesce when it absorbs electromagnetic radiation. Fluorescence and phosphorescence are types of photoluminescence.
- “Chemiluminescent” processes entail the creation of luminescent species by chemical transfer of energy.
- “Electrochemiluminescence” entails creation of luminescent species electrochemically.
- Electrochemiluminescent (ECL) assay techniques are an improvement on chemiluminescent techniques. They provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. In such techniques, the incubated sample is exposed to a voltametric working electrode in order to trigger luminescence. In the proper chemical environment, such electrochemiluminescence is triggered by a voltage impressed on the working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte.
- ECL Electrochemiluminescent
- the luminescence arises from electrochemiluminescence (ECL) induced by exposing the label compound, whether bound or unbound to specific binding partners, to a voltametric working electrode.
- ECL electrochemiluminescence
- the ECL reactive mixture is controllably triggered to emit light by a voltage impressed on the working electrode at a particular time and in a particular manner to generate light.
- Oligonucleotide primers bound to a binding moiety or detectable label and an oligonucleotide probe, labeled at its 3' or 3' and 5' ends with either a binding moiety or detectable label, such that both a binding moiety and a detectable label are both present in the mixture of primer and probe.
- This mixture of probe and primer is introduced into a PCR or other primer directed reaction mixture, such that the mixture is complete for reaction on the addition of the modified primer.
- the 3' or 3'5' labeled probes are not incorporated into the PCR product and thus maintain their specificity for hybridization unlike 5' labeled primers.
- Samples from these PCR/hybridizations are then sampled into a suspension of streptavidin beads and placed directly in an ECL analyzer. This rapid sample handling avoids the wash steps involved with typical hybridization assays.
- FIG. 1 is an assay format for rapid one step assay.
- FIG. 2 is an assay for HIV 1 gag, rapid one step assay.
- FIG. 3 is an assay for the cystic fibrosis gene, rapid one step assay.
- FIG. 4 is an assay for synthetic cystic fibrosis genes, demonstrating its specificity.
- FIG. 5 is an assay for cystic fibrosis genes in normal human samples.
- FIG. 6 is an assay for cystic fibrosis genes in human samples from the University of North Carolina.
- a “nucleotide” is one of four bases: adenine, cytosine, guanine, and thymine (DNA) or uracil (RNA), plus a sugar (deoxyribose for DNA, ribose for RNA), plus a phosphate. In order to provide monomers for the DNA polymerization reaction, typically all four of the deoxynucleotide triphosphates are required.
- a nucleotide as defined herein may also include modified bases such as 5-methyl-dCTP and 7-deaza-dGTP used to improve the action of polymerase on templates.
- nucleotide as used herein also includes bases linked to biotin and digoxigenin (Digoxigenin-11-UTP from Boehringer Mannheim, Indianapolis, Ind.), and biotin-21-UTP and amino-7-dUTP (Clontech, Palo Alto, Calif.) which may be incorporated directly into a primer or into a primer extension product during amplification, to provide for selective binding of amplified sequences.
- biotin and digoxigenin Digoxigenin-11-UTP from Boehringer Mannheim, Indianapolis, Ind.
- biotin-21-UTP and amino-7-dUTP Clontech, Palo Alto, Calif.
- oligonucleotide is a sequence formed of at least two nucleotides.
- a “polynucleotide” is a long oligonucleotide and may be either RNA or DNA. While the term oligonucleotide is generally used in the art to denote smaller nucleic acid chains, and “polynucleotide” is generally used in the art to denote larger nucleic acid chains including DNA or RNA chromosomes or fragments thereof, the use of one or the other term herein is not a limitation or description of size unless expressly stated to be.
- nucleic acid refers to a polynucleotide of any length, including DNA or RNA chromosomes or fragments thereof with or without modified bases as described above.
- sequence e.g. sequence, genetic sequence, polynucleotide sequence, nucleic acid sequence
- sequence refers to the actual enumerated bases (ribose or deoxyribose) present in a polynucleotide strand reading from the 5' to 3' direction.
- the "complement" to a first nucleotide sequence is well known to be a second sequence comprising those bases which will pair by Watson-Crick hybridization with the first sequence.
- the complement to the deoxyribonucleic acid (DNA) sequence 5'-ATGC 3' is well known to be 5'-GCAT 3'(SEQ ID NO: 2).
- DNA deoxyribonucleic acid
- SEQ ID NO: 1 is well known to be 5'-GCAT 3'(SEQ ID NO: 2).
- each of the two strands are described as complementary to the other or as a complementary pair.
- the terms complement and anticomplement may also be used.
- the transcription strand is generally described as plus and its complement as minus (or "+” and “-”), or the transcription strand may be described as the sense strand, and its complement as antisense.
- Two strands each hybridized to the other having all base pairs complementary, are 100% complementary to each other.
- Two strands, each hybridized to the other, having 5% of bases non-complementary, are 95% complementary (or the two strands have 95% complementarily).
- Homology between polynucleotide sequences refers to the degree of sequence similarity between the respective sequences. Two strands which are identical in sequence have 100% sequence homology. Two strands which differ by 5% of sequences have 95% sequence homology. The greater the degree of homology between two strands A and B, the greater the complementarity between A and the complement of B.
- a “probe” is a single or double stranded nucleic acid which has a sequence complementary to a target nucleic acid sequence of interest and which has some additional feature enabling the measurement of the probe-target duplex.
- the artisan will understand that if the probe and/or the target is double stranded, the double stranded nucleic acid must undergo strand separation before hybridization can take place.
- a probe is rendered detectable by an attached tag or label.
- a tag or label linked to a probe may include, in principle, a fluorescent or luminescent tag, an isotopic (e.g. radioisotope or magnetic resonance) label, a dye label, an enzyme label, an antigenic determinant detectable by an antibody, or a binding moiety such as biotin enabling yet another moiety such as a streptavidin coated bead to specifically attach to the probe.
- a “primer” is a relatively short segment of oligonucleotide which is complementary to a portion of the sequence of interest (the sequence of interest can be a subfragment within a larger nucleic acid sequence).
- a primer represents a 5' terminus of the resulting extension product.
- a primer which is complementary at its 3' terminus to the sequence of interest on the template strand enables this 3' terminus to be acted on by a polymerase on hybridization to the template.
- a primer may also be modified at its 5' end with a binding moiety or detectable label.
- “Strand separation” refers to the conversion of a double stranded or duplex nucleic acid to two complementary single stranded polynucleotides.
- the separation process may employ well known techniques including: enzyme mediated separation (e.g. by the enzyme helicase (5), physical-chemical separation (pH, ionic concentration and the like), and thermal separation also known as thermal denaturing.
- Thermal denaturing also referred to as “melting” is the separation of a double stranded polynucleotide (fully or partially duplex) into at least two single strands of polynucleotide by raising the temperature of the solution holding that polynucleotide.
- Hybridization describes the formation of double stranded or duplex nucleic acid from complementary single stranded nucleic acids. Hybridization may take place between sufficiently complementary single stranded DNA and/or RNA to form: DNA-DNA, DNA-RNA, or RNA-RNA.
- DNA polymerase The in vitro amplification of DNA is catalyzed by DNA polymerase.
- a number of types of DNA polymerase are known to the art. They generally share the common property of catalyzing the synthesis of a double stranded DNA sequence utilizing a single stranded template to which a primer is annealed.
- DNA polymerases extracted from most organisms become inactive at the temperatures required for thermal denaturing of nucleic acids. Thus, replacement of the enzyme at the start of each thermal cycle, or the addition of a factor able to prevent heat inactivation, is required if such heat sensitive enzymes are utilized.
- the DNA polymerases which are preferred for in vitro PCR as well as for the invention are derived from organisms which thrive at high temperatures and thus are heat resistant, i.e. thus maintain adequate catalytic activity at the temperature which denatures duplex DNA.
- the reaction catalyzed by DNA polymerase is known to the art, and referred to herein as the "DNA polymerase reaction".
- the reaction requires some or all of the four deoxyribonucleotide triphosphates, primers, preferably in molar excess and a means for cyclic strand separation. Strand separation is preferably achieved by thermal cycling between annealing and denaturation temperatures.
- Reverse transcriptase is known to mediate both RNA to DNA copying, as well as DNA to DNA copying. Hence, any number of enzymes now known will catalyze the chain reaction.
- ECL labels are those which can be made to become luminescent species when acted on electrochemically. Such ECL labels are described in PCT published applications by Bard et al. and Massey et al. (PCT US85/02153, WO86/02734 and PCT US87/00987, WO87/06706).
- ECL apparatus an “ECL analyzer” is any apparatus for performing electrochemiluminescence based assays.
- Oligonucleotides having 3' or 3'5' electrochemiluminescent labels are not able to act as primers in the PCR or other primer directed reactions. They remain at the end of the amplification process available for hybridization to the excess of amplified nucleic acid.
- the invention comprises a method of detecting a nucleic acid sequence of interest in the product of a polymerase chain reaction or other primer directed amplification reaction comprising the steps of (a) incorporating in a polymerase chain reaction or other primer directed reaction mixture, at least one labeled nucleic acid sequence which is capable of electrochemiluminescence and is complementary to said nucleic acid sequence of interest, having the formula: ##STR1## wherein M is ruthenium, osmium or rhenium, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 may be the same or different and each is H, alkyl of 1-4 carbon atoms or a linker group, NA is said nucleic acid sequence linked to one of the bipyridyl groups at its 3' end or to two bipyridyl groups at its 3° and its 5' ends and n is either 1 or 2; (b) conducting a polymerase or other primer directed reaction; and (c) measuring the electrochemil
- Oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer and functionalized with 5' or 3' amino groups using amino modifiers from Clontech (10).
- the oligonucleotides specific for the HIV gag gene PCR assays were the SK38 primer (ATAATCCACCTATCCCAGTAGGAGAAAT) (SEQ ID NO:3) the SK39 primer (TTTGGTCCTTGTCTTATGTCCAGAATGC) (SEQ ID NO:4) and the SK19 probe (ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC) (SEQ ID NO:5) as previously described (6).
- Probe SK19 was labeled at both 3' and 5' ends using the tag-NHS.
- the oligonucleotides were CFF(GACTTCACTTCTAATGATGA) (SEQ ID NO:6) and CFR(CTCTTCTAGTTGGCATGCT) (SEQ ID NO:7) for priming.
- the probes were CFN2 (GAAACACCAAAGATGATATT) (SEQ ID NO:8) for the normal gene and CFD2 (AACACCAATGATATTTTCTTT) (SEQ ID NO:9) for the 508 deletion. These were labeled at 3' and 3'5' sites via amino groups with the N-hydroxy succinimide ester of Ru(bpy) 3 2+ (8,9,11).
- Primers SK39 and CFF were the 5' biotinylated primers in the amplification reaction with unlabeled SK38 and CFR respectively.
- a non-specific oligonucleotide lambda 1 (GAAAATGTGCTGACCGGACATGAAAATGAG) (SEQ ID NO:10) was also synthesized and labeled at the 3' end to use as a control for background signals. Oligonucleotides were prepared for labeling by Biogel P6 column chromatography in 0.3M NaCl, followed by precipitation of the excluded oligonucleotide peak.
- oligonucleotide typically 0.1 ⁇ mole of oligonucleotide was reacted with 0.5 ⁇ mole of ORIGENTM (ruthenium ECL label) Label in 80% dimethyl sulfoxide/phosphate buffered saline, pH 7.4. Biotinylation of the oligonucleotides was performed essentially as above except biotin X-NHS (Clontech) in 50% dimethyl sulfoxide was used for labeling. The labeled oligonucleotides were precipitated with ethanol and washed to remove unincorporated label.
- ORIGENTM ruthenium ECL label
- the synthetic cystic fibrosis sequences used to test the specificity of the system were as follows, the normal sequence, CF normal; GACTTCACTTCTAATGATGATAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACAGAAGCGAGCATGCCAACTAGAAGAG (SEQ ID NO:11) and the mutant sequence, CF D508; GACTTCACTTCTAATGATGATAAAGAAAATATCATTGGTGTTTCCTATGATGAATATAGATACAGAAGCGAGCATGCCAACTAGAAG AG (SEQ ID NO:12).
- the PCR for the HIV1 gag gene was performed essentially as described (6) using a 25 ⁇ l reaction volume containing 75 ng (7.5 pmoles) of the biotinlylated SK39 and 75 ng (7.5 pmoles) of SK38 and 1.25 ng of SK19.
- the temperature cycles used on the Perkin Elmer Thermocycler were as follows: 95° C. 1 min., 60° C. 1 min., the cycle number was 40. This was followed by a cycle of 60° C. 30 min.
- the PCR reaction was run using a biotinylated primer and an unlabeled primer.
- the PCR thermocycles were run as normal for 40 cycles. At the end of the thermocycling an extended incubation was added for the hybridization. The samples at the end of the final incubation cycle were then ready for binding to beads, samples were taken and added to beads for binding (15 min.) at room temperature on an ECL analyzer, then analyzed for electrochemiluminescence. The results are shown in FIG. 1.
- the PCR was run for 35 cycles with the HIV1 gag specific primers including the SK19 3'5' labeled probe.
- the samples of positive HIV1 DNA were dilutions of the standard provided in the Perkin Elmer Cetus Kit. Samples of the PCR 2 ⁇ l were added to streptavidin beads incubated with shaking for 15 min. and analyzed for electrochemiluminescence using an ECL analyzer.
- FIG. 2 demonstrates a simplified PCR assay for rapid detection of the HIV1 gag gene. This ability is due in part to the ECL system and the ECL-based assay formats.
- the ECL system specifically provides (i) a stable label, able to withstand the rigors of the PCR, (ii) an assay modality which due to the nature of the ECL removes the need for external washes, (iii) a sensitivity for the detection of analyte which rivals radioactivity.
- the assay of the invention was used to discriminate genes which had few mutations compared to the normal gene as is the case for the 508 codon deletion in the cystic fibrosis gene.
- the assay was tested using human DNA from cell lines, from human placenta, from normal subjects and with synthetic genes.
- the PCR was run for 30 cycles with the cystic fibrosis specific primers including the 3'5' labeled CFN2.
- the samples of DNA were dilutions of DNA isolated from a human cell line (HeLa,8). Samples of the PCR 2 ⁇ l were added to streptavidin beads incubated with shaking for 15 min. and analyzed for electrochemiluminescence using an ECL analyzer.
- cystic fibrosis gene two synthetic sequences were generated which contained, respectively, the normal gene sequence and the mutant gene sequence, containing the 508 deletion. These gene standards were diluted to the concentration found in human DNA to determine the specificity of the hybridization reaction without any doubt as to the nature of the cystic fibrosis sequences being amplified.
- the PCR was run for 30 cycles with the cystic fibrosis specific primers including the 3' labeled probes CFN2 and CFD2.
- the samples of DNA were dilutions of synthetic DNA made into salmon sperm DNA. The concentration of these sequences was at the same level, as found in human DNA.
- Samples of the PCR 2 ⁇ l were added to 15 ⁇ g of streptavidin beads incubated with shaking for 15 min. and analyzed for electrochemiluminescence using an ECL analyzer.
- results shown in FIG. 4 show the assay's ability to detect specifically the normal or the mutant gene. This rapid assay format is able to rapidly detect and discriminate closely related sequences.
- the PCR was run for 30 cycles with the cystic fibrosis specific primers including the 3'5' labeled probes CFN2 and CFD2.
- the samples of DNA were individual samples of Human DNA isolated from individual chorionic membranes (Sigma Ltd). Samples of the PCR 2 ⁇ l were added to 15 ⁇ g of streptavidin beads incubated with shaking for 15 min. and analyzed for electrochemiluminescence using an ECL analyzer.
- Example IV The data from Example IV was corroborated by assaying a set of human DNA samples from North Carolina where each of the samples was accurately assayed using the method of the invention compared to previous methods (7).
- the data exhibited a wide range of signals due to the vagaries of the PCR and the variation in the sample DNA concentration but with the consistence of the background it was possible to score the presence of the normal gene by the signal obtained with the CFN2 probe and the presence of the mutant gene with the CFD2 probe.
- Sample 15 in these studies gave the lowest signal but still significantly above the non-specific probes signal.
- the data are set forth in FIG. 6.
- the PCR was run for 30 cycles with the cystic fibrosis specific primers including the 3' labeled probes CFN2, CFD2, and a non specific probe (lambda 1).
- the samples of DNA were individual samples of Human DNA at 0.5 to 2.5 ⁇ g/ ⁇ l, 1 ⁇ l of each of these was used in the PCR.
- Samples of the PCR 2 ⁇ l were added to 15 ⁇ g of streptavidin beads incubated with shaking for 15 min. and analyzed for electrochemiluminescence using an ECL analyzer.
Abstract
Description
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 12 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGC4 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCAT4 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATAATCCACCTATCCCAGTAGGAGAAAT28 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TTTGGTCCTTGTCTTATGTCCAGAATGC28 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC41 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GACTTCACTTCTAATGATGA20 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTCTTCTAGTTGGCATGCT19 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAAACACCAAAGATGATATT20 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AACACCAATGATATTTTCTTT21 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GAAAATGTGCTGACCGGACATGAAAATGAG30 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 92 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GACTTCACTTCTAATGATGATAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATAT60 AGATACAGAAGCGAGCATGCCAACTAGAAGAG92 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 89 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GACTTCACTTCTAATGATGATAAAGAAAATATCATTGGTGTTTCCTATGATGAATATAGA60 TACAGAAGCGAGCATGCCAACTAGAAGAG89 __________________________________________________________________________
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US5849544A (en) * | 1992-07-24 | 1998-12-15 | University Of Australia | Amplification and detection process |
US6063568A (en) * | 1996-05-02 | 2000-05-16 | Molecular Innovations, Inc. | Quantitation of RNA transcripts using genomic DNA as the internal amplification competitor |
US6214552B1 (en) | 1998-09-17 | 2001-04-10 | Igen International, Inc. | Assays for measuring nucleic acid damaging activities |
US6280930B1 (en) * | 1996-03-11 | 2001-08-28 | Johnson & Johnson Clinical Diagnostics, Inc. | Amplifying and detecting target nucleic acids using a post amplification incubation step |
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